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Mutational analysis to identify the residues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum

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Abstract

N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of l-arginine that is inhibited by its end product l-arginine in Corynebacterium glutamicum (C. glutamicum). In this study, the potential binding sites of arginine and the residues essential for its inhibition were identified by homology modeling, inhibitor docking, and site-directed mutagenesis. The allosteric inhibition of NAGK was successfully alleviated by a mutation, as determined through analysis of mutant enzymes, which were overexpressed in vivo in C. glutamicum ATCC14067. Analysis of the mutant enzymes and docking analysis demonstrated that residue W23 positions an arginine molecule, and the interaction between arginine and residues L282, L283, and T284 may play an important role in the remote inhibitory process. Based on the results of the docking analysis of the effective mutants, we propose a linkage mechanism for the remote allosteric regulation of NAGK activity, in which residue R209 may play an essential role. In this study, the structure of the arginine-binding site of C. glutamicum NAGK (CgNAGK) was successfully predicted and the roles of the relevant residues were identified, providing new insight into the allosteric regulation of CgNAGK activity and a solid platform for the future construction of an optimized l-arginine producing strain.

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Acknowledgments

I would like to acknowledge Professors Zheng and Lin and Dr. Liang for the encouragement and guidance in this study. The authors are grateful for the financial support of the National Natural Science Foundation of China (31171633).

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Authors and Affiliations

  1. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, People’s Republic of China

    Yuanyuan Huang, Hao Zhang, Hongming Tian, Cheng Li, Shuangyan Han, Ying Lin & Suiping Zheng

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  1. Yuanyuan Huang
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  2. Hao Zhang
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  3. Hongming Tian
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Correspondence to Suiping Zheng.

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Huang, Y., Zhang, H., Tian, H. et al. Mutational analysis to identify the residues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum . Appl Microbiol Biotechnol 99, 7527–7537 (2015). https://doi.org/10.1007/s00253-015-6469-5

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